Oxygen-generating compositions for enhancing cell and tissue survival in vivo

ABSTRACT

A method of treating hypoxic tissue such as wound tissue comprises contacting a composition to the hypoxic tissue in a hypoxia-treatment effective amount, the composition comprising a biodegradable polymer and an inorganic peroxide incorporated into the polymer.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/532,520, which is the 35 U.S.C. § 371 national phase application ofInternational Application No. PCT/US2008/004502, filed Apr. 8, 2008, andpublished in English on Oct. 16, 2008, as International Publication No.WO 2008/124126, and which claims the benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Patent Application Ser. No. 60/910,686, filed Apr. 9,2007, the disclosure of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention concerns methods and materials for treatinghypoxic tissue in vitro and in vivo.

BACKGROUND OF THE INVENTION

Tissue engineering involves assembling cells and their supportstructures together to restore normal function of diseased or damagedtissue (Langer, R. & Vacanti, J. P. Tissue Engineering. Science 260,920-926 (1993)). Supplying sufficient oxygen to the engineered tissue isessential for survival and integration of transplanted cells otherwisenecrosis occurs. However, limitations of oxygen diffusion has led to ageneral conception that cell or tissue components may not be implantedin large volumes (Folkman, J. & Hochberg, M. J Exp Med 138, 745-53(1973)).

Numerous efforts have been made to overcome this limitation, whichinclude the use of oxygen rich fluids such as perfluorocarbons andsilicone oils (Radisic, M. et al. Biomimetic approach to cardiac tissueengineering: Oxygen carriers and channeled scaffolds. Tissue Engineering12, 2077-2091 (2006); Leung, R., Poncelet, D. & Neufeld, R. J.Enhancement of oxygen transfer rate using microencapsulated siliconeoils as oxygen carriers. Journal of Chemical Technology andBiotechnology 68, 37-46 (1997)).

Other approaches to maintaining tissue viability attempted include theuse of angiogenic factors, such as vascular endothelial growth factors(VEGF) and endothelial cells, and cell-support matrices that permitenhanced diffusion across the entire implant (De Coppi, P. et al. TissueEng 11, 1034-44 (2005); Kaigler, D. et al., J Bone Miner Res 21, 735-44(2006); Nomi, M. et al., J. Natl. Cancer Inst. 93, 266-267 (2001).

However, the use of oxygen rich fluids and angiogeneic factors have onlypartially succeeded in achieving survival of a clinically applicablelarge tissue mass.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of treating hypoxictissue in need thereof, comprising contacting a composition to thehypoxic tissue in a hypoxia-treatment effective amount, the compositioncomprising a biodegradable polymer and an inorganic peroxideincorporated into the polymer, preferably in solid form (and optionallya radical trap or decomposing catalyst incorporated into and/or onto thepolymer in solid form).

In some embodiments the hypoxic tissue is in vivo in a subject in needof the treatment.

In some embodiments, the tissue is wound tissue and the composition isadministered in an amount effective to facilitate the healing of thewound tissue.

In some embodiments, the method further comprises the step ofconcurrently treating the wound tissue with negative pressure woundtherapy.

In some embodiments, the tissue is afflicted with an anaerobic infectionand the composition is administered in an amount effective to treat theinfection.

In some embodiments, the tissue is cancer tissue and the composition isadministered in an amount effective to treat the cancer (alone, or incombination with one or more additional therapeutic agents).

In some embodiments, the composition is in the form of a sheet material,and the contacting step is carried out by contacting the sheet materialto the tissue. In some embodiments, the composition is in the form ofinjectable microparticles, and the contacting step is carried out byinjecting the microparticles into the tissue. In some embodiments, thecomposition is in the form of a spray, and the contacting step iscarried out by spraying the composition onto the tissue. In someembodiments, the composition is in the form of a surgical or paramedicalaid, and the contacting step is carried out by contacting the aid to thetissue.

A second aspect of the invention is a composition comprising, consistingof, or consisting essentially of;

(a) from 50 or 70 to 99 percent by weight of a biodegradable polymer;and

(b) from 0.1 to 30 percent by weight of inorganic peroxide incorporatedinto the polymer in solid form; and

(c) optionally from 0.1 to 30 percent by weight of a radical trap orperoxide decomposition catalyst incorporated into the polymer in solidform; and

(d) optionally from 0.001 to 5 percent by weight of at least oneadditional active agent (e.g., antibiotics, growth factors, steroids,antineoplastic agents, etc.). The composition may be in the form ofsheet material, injectable microparticles, other shaped articles orscaffolds, etc.

A still further aspect of the invention is, in a method of culturingmammalian tissue in vitro on a solid support or scaffold, theimprovement comprising utilizing as the scaffold a compositioncomprising a biodegradable polymer and an inorganic peroxideincorporated into the polymer in solid form so that oxygenation of thetissue is thereby enhanced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Oxygen release from POG film, in vitro. In the formulation usedthe release of oxygen follows a sigmoidal curve with a high rate ofoxygen release during the first 24 hours. The control material, PLGA,did not show any oxygen release.

FIG. 2: Flap Necrosis. Graph expressing the necrosis in percent of totalflap size. At early time points, 2 and 3 days, the SPO group showed asignificant better flap survival with less necrosis, when compared tothe control group. However, after 7 days the area of necrosis wassimilar in both groups.

FIG. 3: Histological Analysis, 100×: Hematoxylin and Eosin stains of theskin flaps harvested at 3 and 7 days showed delayed necrosis in the SPOgroup with better conservation of tissue architecture, epidermis height,hair follicles and sebaceous glands. Differences were more prominent atthe 7 day time point.

FIG. 4: Evaluation of Apoptosis after 3 days: a, representative sectionsof dermis showing apoptosis positive cells with brown nuclei (nucleicounterstained with methyl green). b, A significant higher number ofapoptotic cells were found in the dermis of the control (PLGA) groupwhen compared with the treatment group (SPO). The oxygen generatingbiomaterial was able to prevent or delay the induction of apoptosis inthe skin flap.

FIG. 5: Tissue Lactate Levels: Graph presenting the flap tissue lactatelevels corrected for tissue weight. Individual dots are presenting eachanimal and bars indicate the average of the group. High level of lactateis an indication of low oxidative metabolism due to low oxygen tensionin tissues. The SPO group shows better results with lower levels oflactate, when compared to PLGA only.

FIG. 6: Biomechanical Testing. Graph showing the maximal tensile strainat break in MPa. In average, the SPO group showed a higher maximaltensile strain at the 3 day time point with no statistical differencewhen compared to normal skin. This indicates that the SPO was able todelay the breakdown of proteins in the extracellular matrix of thedermis. At the 7 day the strains of the two groups were comparable.

FIG. 7: Enhanced cell viability is observed when 3T3 cells incorporatedinto an approximately 1 cm cube PLGA scaffold containing calciumperoxide (CPO) are incubated under extend hypoxic (<1% oxygen)conditions. The control contains no oxygen producing materials.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Subjects that can be treated by the methods and compositions of thepresent invention include both human subjects and animal subjects(including but not limited to other mammalian subjects such as dog, cat,horse, cow, sheep, rabbit, goat, pig, monkey, etc.).

The disclosures of all patent references cited herein are to beincorporated herein by reference in their entirety.

A. Compositions.

Any suitable biodegradable polymer can be used to carry out the presentinvention, including but not limited to poly(lactide)s,poly(glycolide)s, poly(lactide-coglycolide)s, poly(lactic acid)s,poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s,poly(caprolactone), polycarbonates, polyesteramides, polyanhydrides,poly(amino acid)s, poly(ortho ester)s, polycyanoacrylates, polyamides,polyacetals, poly(ether ester)s, copolymers of poly(ethylene glycol) andpoly(ortho ester)s, poly(dioxanone)s, poly(allcylene alkylate)s,biodegradable polyurethanes, as well as blends and copolymers thereof.See, e.g., U.S. Pat. No. 7,097,857; see also U.S. Pat. Nos. 6,991,652and 6,969,480.

The oxygen-generating active agent is preferably an organic or inorganicperoxide such as urea peroxide, calcium peroxide, and magnesiumperoxide, and sodium percarbonate. The oxygen-generating active agent isincluded in the composition in any suitable amount (e.g., from 0.1 or 1to 10, 20, or 30 percent by weight, or more). In some embodimentscalcium peroxide is preferred as it releases oxygen at a desireable ratein situ. The oxygen-generating active agent can be included in thepolymer in solid form, such as in the form of a plurality of solidparticles thereof.

In some embodiments a radical trap or peroxide or radical decompositioncatalyst is also included in the composition (e.g., in an amount of from0.1 or 1 to 10, 20 or 30 percent by weight, or more). Suitable examplesof radical traps or decomposition catalysts include, but are not limitedto, iron (including, but not limited to, iron particles ornanoparticles, enzymes such as catalase, peroxidase; or dehydrogenase(see, e.g., U.S. Pat. No. 7,189,329), compounds such as cyclicsalen-metal compounds that have superoxide and/or catalase and/orperoxidase activity (see, e.g., U.S. Pat. No. 7,122,537), etc.). Theradical trap or decomposing catalyst may be included in solid form(e.g., solid particulate form) and can be coated on or incorporated inthe polymer, or both coated on and incorporated in the polymer).

Sheet materials can be formed of the polymer and the oxygen-generatingactive agent by any suitable technique, including but not limited todipping, spraying, casting, extruding, etc.

Injectable microparticles and methods of making the same are known anddescribed in, for example, U.S. Pat. Nos. 7,101,568; 6,455,526;6,350,464; 5,482,927; 4,542,025; and 4,530,840. Injectablemicroparticles may be of any suitable size and shape, for example havingan average diameter of from 1, 3 or 5 micrometers, up to 300, 500 or 700micrometers.

In some embodiments (e.g., for use as tissue scaffolds) the compositionsare formed into articles such as sheet materials or other formedarticles that have a thickness of at least 100 micrometers(approximately the diffusion distance of oxygen in tissue). Irrespectiveof their shape (e.g., particulate, sheet, or other formed article), thecompositions may be solid or porous as desired for the intented use.

The compositions, in the form of sheets, microparticles, or any othersuitable form, can be packaged in sterile form in a sterile containerfor subsequent use.

Injectable microparticles can be provided and injected “dry” or combinedwith a sterile physiologically acceptable liquid carrier such asphysiological saline solution for injection.

In some embodiments, the composition may contain one or more additionalactive agents (e.g., from 0.0001 or 0.001 to 1, 5 or 10 percent byweight). Examples of such additional active agents include, but are notlimited to, chemotherapeutic agents, herbicides, growth inhibitors,anti-fungal agents, anti-bacterial agents, anti-viral agents andanti-parasitic agents, mycoplasma treatments, growth factors, steroids,proteins, nucleic acids, angiogenic factors, anaesthetics,mucopolysaccharides, metals, wound healing agents, growth promoters,indicators of change in the environment, enzymes, nutrients, vitamins,minerals, carbohydrates, fats, fatty acids, nucleosides, nucleotides,amino acids, sera, antibodies and fragments thereof, lectins, immunestimulants, immune suppressors, coagulation factors, neurochemicals,cellular receptors, antigens, adjuvants, radioactive materials, andother agents that effect cells or cellular processes. See, e.g., B.Gibbins et al., US Patent Application Pub. No. 2001/0041188.

“Antibiotic” as used herein may be any suitable antibiotic, includingbut not limited to Amikacin, Gentamicin, Spectinomycin, Tobramycin,Imipenem, Meropenem, Cefadroxil, Cefazolin, Cephalexin, Cefaclor,Cefotetan, Cefoxitin, Cefprozil, Cefuroxime, Loracarbef, Cefdinir,Cefixime, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime,Ceftibuten, Ceftozoxime, Ceftriaxone, Cefepime, Azithromycin,Clarithromycin, Dirithromycin, Penicillin G, Cloxacillin, Dicloxacillin,Nafcillin, Oxacillin, Amoxicillin, Ampicillin, Mezlocillin,Piperacillin, Nalidixic Acid, Ciprofloxacin, Enoxacin, Lomefloxacin,Norfloxacin, Ofloxacin, Levofloxacin, Sparfloxacin, Alatrofloxacin,Gatifloxacin, Moxifloxacin, Trimethoprim, Sulfisoxazole,Sulfamethoxazole, Doxycycline, Minocycline, Tetracycline, Aztreonam,Chloramphenicol, Clindamycin, Quinupristin, Fosfomycin, Metronidazole,Nitrofurantoin, Rifampin, Trimethoprim, and Vancomycin. See, e.g., U.S.Pat. No. 6,605,609. Antibiotics suitable for use against anaerobicanaerobic bacteria include, but are not limited to, chloramphenicol,metronidazole, imipenem, clindamycin and cefoxitin.

“Growth factor” as used herein basic fibroblast growth factor (bFGF),acidic fibroblast growth factor (aFGF), nerve growth factor (NGF),epidermal growth factor (EGF), insulin-like growth factors 1 and 2,(IGF-1 and IGF-2), platelet derived growth factor (PDGF), tumorangiogenesis factor (TAF), vascular endothelial growth factor (VEGF),corticotropin releasing factor (CRF), transforming growth factors alphaand beta (TGF-alpha and TGF-beta), interleukin-8 (IL-8);granulocyte-macrophage colony stimulating factor (GM-CSF); theinterleukins, and the interferons. See, e.g., B. Gibbins et al., USPatent Application Pub. No. 2001/0041188.

“Steroid” as used herein may be any suitable steroid, including but notlimited to those described in U.S. Pat. No. 7,157,433

“Antineoplastic agent” as used herein includes, without limitation,platinum-based agents, such as carboplatin and cisplatin; nitrogenmustard alkylating agents; nitrosourea alkylating agents, such ascarmustine (BCNU) and other alkylating agents; antimetabolites, such asmethotrexate; purine analog antimetabolites; pyrimidine analogantimetabolites, such as fluorouracil (5-FU) and gemcitabine; hormonalantineoplastics, such as goserelin, leuprolide, and tamoxifen; naturalantineoplastics, such as taxanes (e.g., docetaxel and paclitaxel),aldesleukin, interleukin-2, etoposide (VP-16), interferon alfa, andtretinoin (ATRA); antibiotic natural antineoplastics, such as bleomycin,dactinomycin, daunorubicin, doxorubicin, and mitomycin; and vincaalkaloid natural antineoplastics, such as vinblastine and vincristine.See, e.g., U.S. Pat. No. 7,101,568.

“Local active agents” or “topical active agents” (i.e., those for localand/or topical administration) as used herein include, but not limitedto, those agents set forth above, topical antibiotics and otheranti-acne agents, anti-fungal agents, anti-psoriatic agents,antipruritic agents, antihistamines, antineoplastic agents, localanesthetics, anti-inflammatory agents and the like. Suitable topicalantibiotic agents include, but are not limited to, antibiotics of thelincomycin family (referring to a class of antibiotic agents originallyrecovered from streptomyces lincolnensis), antibiotics of thetetracycline family (referring to a class of antibiotic agentsoriginally recovered from streptomyces aureofaciens), and sulfur-basedantibiotics, i.e., sulfonamides. Exemplary antibiotics of the lincomycinfamily include lincomycin itself(6,8-dideoxy-6-[[(1-methyl-4-propyl-2-pyrrolidinyl)-carbonyl]amino]-1-thio-L-threo-.alpha.-D-galacto-octopyranoside),clindamycin, the 7-deoxy, 7-chloro derivative of lincomycin (i.e.,7-chloro-6,7,8-trideoxy-6-[[(1-methyl-4-propyl-2-pyrrolidinyl)carbonyl]-amino]-1-thio-L-threo-.alpha.-D-galacto-octopyranoside),related compounds as described, for example, in U.S. Pat. Nos.3,475,407, 3,509,127, 3,544,551 and 3,513,155, and pharmacologicallyacceptable salts and esters thereof. Exemplary antibiotics of thetetracycline family include tetracycline itself4-(dimethylamino)-1,4,4.alpha.,5,5.alpha.,6,11,12.alpha.-octahydro-3,6,12,12.alpha.-pentahydroxy-6-methyl-1,11-dioxo-2-naphthacene-carboxamide),chlortetracycline, oxytetracycline, tetracycline, demeclocycline,rolitetracycline, methacycline and doxycycline and theirpharmaceutically acceptable salts and esters, particularly acid additionsalts such as the hydrochloride salt. Exemplary sulfur-based antibioticsinclude, but are not limited to, the sulfonamides sulfacetamide,sulfabenzamide, sulfadiazine, sulfadoxine, sulfamerazine,sulfamethazine, sulfamethizole, sulfamethoxazole, and pharmacologicallyacceptable salts and esters thereof, e.g., sulfacetamide sodium. Topicalanti-acne agents include keratolytics such as salicyclic acid, retinoicacid (“Retin-A”), and organic peroxides, while topical antifungal agentsinclude amphotericin B, benzoic acid, butoconazole, caprylic acid,econazole, fluconazole, itraconazole, ketoconazole, miconazole,nystatin, salicylic acid, and terconazole, and topical antipsoriaticagents include anthralin, azathioprine, calcipotriene, calcitriol,colchicine, cyclosporine, retinoids, and vitamin A. The active agent mayalso be a topical corticosteroid, and may be one of the lower potencycorticosteroids such as hydrocortisone, hydrocortisone-21-monoesters(e.g., hydrocortisone-21-acetate, hydrocortisone-21-butyrate,hydrocortisone-21-propionate, hydrocortisone-21-valerate, etc.),hydrocortisone-17, 21-diesters (e.g., hydrocortisone-17,21-diacetate,hydrocortisone-17-acetate-21-butyrate, hydrocortisone-17,21-dibutyrate,etc.), alclometasone, dexamethasone, flumethasone, prednisolone, ormethylprednisolone, or may be a higher potency corticosteroid such asclobetasol propionate, betamethasone benzoate, betamethasonediproprionate, diflorasone diacetate, fluocinonide, mometasone furoate,triamcinolone acetonide, or the like. See, e.g., U.S. Pat. No.6,582,714.

B. Tissue In Vitro and In Vivo.

Compositions of the invention may be used to treat or oxygenate cellsand tissues in vitro by any suitable means, such as by topicallyapplying the composition to the cells (e.g., as a sheet material or asmicroparticles), growing cells on and/or into the compositions (e.g.,compositions in sheet or other solid substrate form), injecting thecompositions into cells or tissues being grown in vitro, etc.

Culturing of mammalian tissues (e.g., dog, cat, mouse, monkey, humantissues, etc.) in vitro is known and can be carried out in accordancewith known techniques. In general culturing is carried out in abioreactor, with suitable cells seeded or deposited on a support orscaffold, which scaffold is in turn placed in a suitable growth orculture media. See, e.g., U.S. Pat. Nos. 4,940,853; 6,592,623;6,645,759; 6,645,759; etc. Examples of suitable tissues for culturinginclude, but are not limited to, bone tissue, skin tissue (e.g., dermaltissue), skeletal muscle, cardiac muscle, vascular (e.g., blood vessel)tissue, and other tissues that are comprised of oxygen-sensitive cells.

In still further embodiments, the compositions as described above can beimplanted in vivo as a tissue scaffold in a suitable subject,particularly in regions where circulation might otherwise be impaired.

C. Wound Treatment.

In some embodiments the region of interest or the tissue being treatedis a wound site or a region adjacent the wound site in a patient orsubject afflicted with that wound.

“Wound” as used herein includes any type of accidental or deliberate(e.g., surgical) tissue trauma, including but not limited to incisions,lacerations, ulcers, abrasions, burns, crush injuries, amputations,punctures, and combinations thereof.

“Wound tissue” is tissue the health of which is deleteriously affectedby a wound (e.g., disrupted circulation as in a skin flap, crush injury,etc.).

In some embodiments the wound or wound tissue is also infected orafflicted with an anaerobic infection as described further herein.

In one embodiment of the invention, the compositions are administered towounds of subjects that are concurrently undergoing treatment withnegative pressure wound therapy. For example, in some embodiments, acomposition of the invention in the form of a sheet material (which mayoptionally contain perforations) is applied to a wound or wound tissuebefore or shortly before a negative pressure chamber is applied over thewound tissue and negative pressure is applied, so that oxygen isadministered from the composition to the afflicted tissue during thenegative pressure wound therapy. In this embodiment, the sheet materialmay optionally serve as the “interface” or “dressing” in the negativepressure wound therapy system. In other embodiments, the composition inthe form of injectable microparticles is topically applied to the woundtissue or injected into the wound tissue (e.g. injected below thesurface of the wound, under the wound, into regions immediately adjacentthe wound to facilitate oxygen transfer into afflicted tissue) before orshortly before a negative pressure chamber is applied over the woundtissue and negative pressure is applied thereto, so that oxygen isadministered from the composition to the afflicted tissue during thenegative pressure wound therapy. Negative pressure wound therapy isknown and describes techniques in which wound healing is facilitated bythe application of a vacuum, or negative pressure, to the wound. See,e.g., U.S. Pat. No. 5,645,081 The specific modality of implementation isnot critical and any of a variety of techniques can be employed,including but not limited to those described in U.S. Pat. Nos.7,004,915; 6,951,553; 6,855,135; 6,800,074; 6,695,823; and 6,458,109.

When used to treat wounds, the composition may optionally contain one ormore additional local or topical active agents to facilitate growth ortreat infection (e.g. an antibiotic), treat pain (e.g., an analgesic),treat inflammation (e.g., an antiinflammatory agent such as anonsteroidal antiinflammatory agent) as described above.

D. Surgical Aids, Paramedic Aids, and Sprays.

Surgical and paramedic aids can be produced from the materials of theinvention. Such aids generally comprise comprise a biodegradable polymerand an inorganic peroxide incorporated into said polymer in solid form,as described above. The aid can be in any suitable form, such as asponge, packing, wound dressing (gauze, adhesive bandage, etc.,),suture, etc. A support material for the biodegradable polymer can beprovided if desired, with the polymer contacted to the support materialin the form of a sheet, powder, particles, etc. The aid can be packagedin sterile form in a suitable container, or provided in non-sterile formfor sterilization by the user.

For some applications the compositions can be provided as spraycompositions, the spray compositions comprising a carrier (e.g., anaqueous or non-aqueous carrier), a biodegradable polymer, and aninorganic peroxide in solid form, as described above. The polymer can besolubilized or dispersed in the carrier (that is, the spray compositionscan be in the form of solutions, suspensions, dispersions,microdispersions, emulsions, etc.). The spray compositions can beprovided in a suitable spray applicator such as a pump-type sprayapplicator, or can be packaged in an aerosol container with a suitablepropellant (e.g., HFA-134a, HFA-227ea, carbon dioxide) to provide anaerosol spray device containing an aerosol spray composition. The spraycompositions may advantageously include additional active agents, all asdescribed above. Such spray compositions can be formulated in accordancewith known techniques or variatious thereof that will be apparent tothose skilled in the art. See, e.g U.S. Pat. Nos. 7,182,277; 7,163,672;7,101,535; 7,090,831; 7,074,388; 6,218,353; etc.

E. Anaerobic Infections.

The compositions of the invention may be used to treat anaerobicinfections in any tissue so infected, for example in a subject afflictedwith such an infection. Anaerobic infections may be caused by any of avariety of anaerobic bacteria, including but not limited to Bacteroidesspecies (e.g., Bacteroides fragilis), Peptostreptococcus, andClostridium species (e.g., Clostridium perfringens).

Examples of anaerobic infections include, but are not limited to, gasgangrene, clostridial myonecrosis, necrotizing infections such asnecrotizing fascitis, etc. Particular examples include anaerobicinfections of the mouth, head, and neck (e.g. infections in root canals,gums (gingivitis), jaw, tonsils, throat, sinuses, ears, etc.); anaerobicinfections of the lung (e.g., as in pneumonia, lung abscesses, infectionof the lining of the lung (empyema), and dilated lung bronchi(bronchiectasis)); anaerobic infections of the abdominal cavity, such asintraabdominal abscess formation, peritonitis, and appendicitis;anaerobic infections of the female genital tract (e.g., pelvicabscesses, pelvic inflammatory disease, inflammation of the uterinelining (endometritis), and pelvic infections following abortion,childbirth, and surgery); anaerobic infections of skin and soft tissue(e.g., diabetic skin ulcers, gangrene, destructive infection of the deepskin and tissues (necrotizing fascitis), and bite wound infections);anaerobic infections of the central nervous system (e.g., cause brainand spinal cord abscesses); bacteremia; etc.

The route of administration will depend upon the particular type ofinfection and the tissue infected. In some embodiments a sheet materialof the composition may be applied in like manner as described inconnection with wounds above. In other embodiments injectablemicroparticles of the composition may be injected as described inconnection with wounds above. Multiple routes of administration (e.g.,both topical and injection) may be used. Surgical removal of damagedtissue and/or drainage of adversely affected areas (e.g., by negativepressure wound treatment or any other means) may also be used. Multipleadministrations may be desired. The compositions may have an additionalactive agent such as one or more antibiotics incorporated therein, asalso described above.

F. Tumor Treatment.

In some embodiments, the compositions of the invention can be used totreat a tumor or cancer in a subject in need thereof by administeringthe compositions to a subject in need thereof. Administration may becarried out by any technique that brings the compositions into contactwith tumor cells, including but not limited to injecting the compositionin microparticle form into the tumor, or into a region containing oradjacent the tumor. Tumors or cancers that can be treated include butare not limited to lung, colon, liver, prostate, breast, ovarian, skin,and pancreatic cancer.

The present invention is explained in greater detail in the non-limitingexamples set forth in the experimental section below.

Experimental

Here we show that implantable oxygen releasing biomaterials can providea sustained release of oxygen to cells and tissues resulting inprolonged tissue survival and decreased necrosis. Both visibly andhistologically, significant decreases in necrosis were observed forleast two days after implantation of the oxygenating biomaterial inmice. Our results show that oxygen producing biomaterials can releaseoxygen into hypoxic tissues resulting in a delayed onset of necrosis.These findings are also applicable to treating cancer, since hypoxia hasbeen shown to promote the aggressiveness of cancer cells (Hockel, M. &Vaupel, P. J. Natl. Cancer Inst. 93, 266-267 (2001)). In addition, thistechnology is also be useful for various wound healing andreconstructive applications including plastic surgery with flaps andtreatment of anaerobic infections (Jonsson, K. et al., Annals of Surgery214, 605-613 (1991); Bowler, P. G. et al., Clinical Microbiology Reviews14, 244 (2001)).

First, we prepared a polymeric oxygen generating (POG) film bydispersing sodium percarbonate (SPO) into poly(lactide-co-glycolide)(PLGA). Sodium percarbonate is an adduct of hydrogen peroxide and sodiumbicarbonate which spontaneously decomposes upon contact with water toproduce oxygen. When the POG films are placed in a moist environment at37° C., oxygen production was observed over a 24 hour period. (FIG. 1).As expected, no oxygen release was detected from the control films ofPLGA. To study how oxygen generating biomaterials can affectcritically/marginal perfused tissues in vivo we used the skin flap modelin mice. This model is well accepted as serves as a standard forresearch on ischemic tissue survival (Buemi, M. et al., Shock 22,169-173 (2004); Giunta, R. E. et al., J. Gene Med 7, 297-306 (2005);Gould, L. J. et al., Wound Repair Regen. 13, 576-582 (2005)). 11-13 Weanalyzed the adjacent tissue flaps on tissue, cellular and biochemicallevels.

After generating the rectangular dorsal skin flaps, an oxygen producingfilm was placed in the subcutaneous space in all animals, and thesurgical wound was closed. PLGA films containing no sodium percarbonatewere used as controls. The size of tissue necrosis was measured at 2, 3and 7 days. The analysis of graft necrosis shows a significant benefitfor the SPO group in the early time points with p=0.001 at 2 days andp=0.025 at 3 days, respectively.

After 7 days however, the size of tissue necrosis were comparablebetween the two groups (p=0.74). (FIG. 2) The prevention of necrosis bythe oxygen releasing biomaterial is a key finding on this study andmight indicate that the early supplementation with oxygen was able todelay tissue death. Characterization at a cellular level involvedhistological examination of tissue sections at 3 and 7 days. Tissuesections were stained with hematoxyin and eosin to explore the extent oftissue necrosis. Overall, there was a clear survival benefit for the POGgroup with better preservation of general tissue architecture, epidermisheight, hair follicles and sebaceous glands. In the early time point,the control group had already lost much of the height of the stratifiedlayer and the dermis. Hair follicle and the glands were less definedwhen compared to the POG group. However, at 3 days the general tissuearchitecture was conserved in both groups including, clearly definedepidermis and dermis separated by basal cells and the basilar membrane.In the control group this picture changed drastically at 7 days withdisruption of tissue architecture and vague transitions between thelayers and an eosin positive mass replacing the dermis. The loss ofdermal papilla, sebaceous glands and hair follicle defined a moreadvanced stage of necrosis. The POG group showed a slower progressionwith remaining defined layers and intact hair follicles. Both groupsshowed a mixed cellular infiltration starting from the flap edge, with atendency for higher cellular content in the dermis of the POG group.

To compare the induction of apoptosis in the flaps of the two groups, weperformed a TUNEL stain (FIG. 3). We found significantly lower amountsof apoptosis positive cells in the dermis of the POG group at 3 days(p=0.030). Due to the more advanced stage of tissue degradation withdisruption of the nuclear envelope at 7 days the TUNEL stain was onlyable to show poorly defined DNA smears. In summary, the histologicalfindings and significant lower level of apoptosis indicate a delayedonset of necrosis in the group with the oxygen releasing biomaterial.

Apoptosis was evaluated in flap tissue treated by the compositions ofthe invention after three days (FIG. 4). FIG. 4a provides representativesections of dermis showing apoptosis positive cells with brown nuclei(nuclei counterstained with methyl green). As shown in FIG. 4b , asignificantly higher number of apoptotic cells were found in the dermisof the control (PLGA) group when compared with the treatment group(SPO). The oxygen generating biomaterial was able to prevent or delaythe induction of apoptosis in the skin flap.

Additional investigations at the biochemical level were conducted bymeasuring the lactate concentration in the flap tissue. Higherconcentrations of lactate are related to various medical conditionsinducing anaerobe metabolism and, therefore, a reliable measure foranaerobic metabolism do to poor vascularization. We found that thetissue flaps with underlying oxygen producing biomaterial had lowerlactate levels when compared to the control group. (FIG. 5) Thedifference was more prominent at the early time point with the POG grouphaving 57% lactate of the control group. The lactate levels in bothgroups increased at the seven day time point. The anticipated increaseafter seven days is countered by the reduction of living cells in thetissue. The lower level of lactate in the POG group is a furtherindication that the oxygen releasing biomaterial is able to supportcellular survival.

To further investigate the impact of the oxygen releasing biomaterial ontissue integrity, we measured wound breaking strengths. (FIG. 6) At timeof harvest longitudinal tissue specimens were cut form the skin flap andpulled to failure. In the early time point skin samples from the controlhad already lost significant strength (p=0.028) when compared to normalskin. The decrease of strength in the POG group was not significant(p=0.130). However, after 7 days both samples were comparable and showedsignificant loss of strength with p=0.008 for the PLGA group andp=−0.004 for the POG group, respectively. Biomechanical testing was ableto show a benefit in tissue integrity for the POG group in the earlytime points. Since degradation of extracellular matrix proteins throughhydrolysis and enzymatic degradation is common during tissue death thehigher tissue strength in the POG group indicates a delayed tissuenecrosis.

As shown in FIG. 7, enhanced cell viability is observed when 3T3 cellsincorporated into an approximately 1 cm cube PLGA scaffold containingcalcium peroxide (CPO) are incubated under extended hypoxic (<1% oxygen)conditions. The control contains no oxygen producing materials. Note inparticular that ten days of data are shown.

Methods

Production of Polymeric Oxygen Generating Films.

The Poly(D,L-lactide-coglycolide) (PLGA 50:50, i.v. 0.89 dl/g in HFIP at30° C., Lactel Absorbable Polymers, Pelham Ala.) films incorporatingsodium percarbonate (SPO, Geel, Belgium) or calcium peroxide (CPO) werefabricated using a solvent casting process from mixtures of PLGA andSPO. Briefly, PLGA was dissolved in methylene chloride (5% w/v) and SPOwas pulverized by freezer-mill (SPEX 6700, Mutchen, N.J.). PLGA solutionand SPO were thoroughly mixed using a vortex. Films were cast from PLGAsolution on Pyrex glass dish (Φ100) containing SPO in specifiedconcentrations. In order to prevent the formation of voids within thepolymer films a glass cover was put on the moulds to provide slowsolvent evaporation. After 48 hrs of solvent evaporation, the films weredried for 72 hrs in a drying chamber under vacuum condition at roomtemperature. PLGA film without SPO was used as a control, and wasfabricated using the same method. All other chemicals of analyticalgrade were purchased from Sigma (St. Louis, Mo.). Oxygen release wasmeasured by recording the displaced volume of water displaced with thecollected oxygen gas generated.

Animal Model:

16 nude mice (Nude-nude, Charles River Laboratories Inc. Wilmington,Mass.) were randomized into 2 groups. Group 1 received the oxygenreleasing biomaterial (SPO) while Group 2 served as the control groupand received the PLGA only biomaterial (PLGA). Four animals of eachgroup were sacrificed on day 3 and day 7. All procedures were performedin accordance with the Animal care and use committee. We used theestablished skin flap model in mice to produce critically vascularizedskin flaps. All surgeries were performed under general anesthesia usingisoflurane 2%. Skin flaps 30×10 mm in size were created on the back. Theu-shaped flap reduced the intact vascularity to the 1 cm wide base. Inboth groups a 20×10 mm large biomaterial was placed subcutaneous betweenmuscle and skin layer. The surgical wound was then closed usingabsorbable suture material in a running fashion. All animals survivedthe surgeries without complications. During the first 24 h the micereceived routine analgesic injection with buprenorphine 0.1 mg/kg 3times per day. The animals were allowed free access to food and waterand housed in a 12 hour day/night cycle. At time of sacrifice theanimals were euthanized by CO₂. For consistency tissue samples for allthe individual tests were taken in a standard fashion.

Assessment of Graft Necrosis.

At day 2, 3 and 7 all animals were anesthetized by isoflurane 2% andphotographed under standard lighting. The necrosis was clearly visiblewith a change in skin color towards brown/black. The flap necrosis wasmeasured by image analysis (Image J, NIH) and expressed in percent oftotal flap size.

Histology.

Skin samples harvested at 3 and 7 days were embedded in Tissue-Tek®O.C.T. Compound 4583 (Sakura®), frozen in liquid nitrogen and sectionedinto 6-8 μm slices using a cryostat (CM 1850 Cryotsat, Leica,Bannockburn, Ill.). Tissue samples of each animal were stained withhematoxylin and eosin using standard protocols. Histology was used toassess the level of inflammation, remaining tissue architecture andnecrosis. Further, TUNEL staining (TACS TdT Kit, R&D Systems,Minneapolis, Minn.) was performed according to the manufacturesguidelines to investigate the level of induced apoptosis within theepidermis of the skin flaps. Images were taken using a Zeiss Axio ImagerM1 Microscope (Carl Zeiss, Thornwood, N.Y.). Image analysis wasperformed using Image J (NIH) Image analysis Software. Number ofapoptotic cells per high power field (400×) was counted and comparedbetween the groups.

Lactate Assay.

Tissue samples were stored at −80° C. and homogenized using cryogrinding. The powder was weighed and the tissue particles furtherdissolved in cold 3M HClO₄ (0.15 ml per vial) of for 20 Minutes. Alltubes were handled in an ice/salt bath at −8° C. Following acentrifugation at 11,500 RPM for 10 minutes at 4° C. the supernatant wascollected and tissue L-lactate determined spectrophotometrically using acommercially available kit (Lactate Assay Kit, BioVision Research,Mountain View, Calif.). Briefly, samples were transferred to a 96 welldish containing enzyme and reaction buffer. After 30 minutes at roomtemperature the colorimetric change was measured by aspectrophotometrically at 540 nm.

Biomechanical Testing.

Rectangular longitudinal tissue strips (30 mm×5 mm) were used toevaluate the would-braking strength. Tensile tests (Instron model 5544,Issaquah, Wash., USA) were performed by elongating the tissue stripslongitudinally at a speed of 0.05 mm/second with a preload of 0.2 Nuntil failure. The grip-to-grip spacing was 2 cm. All specimens weretested at room temperature and kept moist. The maximum tensile strain(MPa) was determined.

Statistics.

All presented data is expressed as averages and the correspondingstandard deviations. For statistical analysis we used SPSS v11 (SPSSInc). Differences between the two groups were analyzed by 2 tailedindependent samples T test. For the analysis of the biomechanicalproperties we used one-way analysis of variance (ANOVA) followed by aBonferroni test for multiple comparisons. A p value of less then 0.05was considered significant.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of treating hypoxic tissue in needthereof, comprising: contacting a composition to said hypoxic tissue ina hypoxia-treatment effective amount, wherein said composition comprisesa biodegradable polymeric film and an inorganic peroxide incorporatedinto said polymeric film, wherein said inorganic peroxide comprises aplurality of solid inorganic peroxide particles, and wherein saidhypoxic tissue is wound tissue, tissue afflicted with an anaerobicinfection or cancer tissue.
 2. The method of claim 1, wherein saidhypoxic tissue is in vivo in a subject in need of said treatment.
 3. Themethod of claim 1, wherein said tissue is wound tissue and saidcomposition is administered in an amount effective to facilitate thehealing of said wound tissue.
 4. The method of claim 2, furthercomprising the step of concurrently treating said wound tissue withnegative pressure wound therapy.
 5. The method of claim 1, wherein saidtissue is afflicted with an anaerobic infection and said composition isadministered in an amount effective to treat said infection.
 6. Themethod of claim 1, wherein said tissue is cancer tissue and saidcomposition is administered in an amount effective to treat said cancer.7. The method of claim 1, wherein said composition is in the form of asheet material, and said contacting step is carried out by contactingsaid sheet material to said tissue.
 8. The method of claim 1, whereinsaid composition is in the form of a surgical or paramedical aid, andsaid contacting step is carried out by contacting said aid to saidtissue.
 9. The method of claim 1, wherein said biodegradable polymericfilm comprises a polymer selected from the group consisting ofpolylactide, polyglycolide, poly lactide-glycolide copolymers, andalginate.
 10. The method of claim 1, wherein said peroxide is calciumperoxide.
 11. The method of claim 1, wherein said peroxide is sodiumpercarbonate.
 12. The method of claim 1, wherein said compositioncomprises: (a) from 70 to 99 percent by weight of the biodegradablepolymeric film; and (b) from 1 to 30 percent by weight of the inorganicperoxide incorporated into said polymeric film; (c) optionally from 0.1to 30 percent by weight of a radical trap or peroxide decompositioncatalyst incorporated into the polymeric film in solid form; and (d)optionally from 0.001 to 5 percent by weight of at least one additionalactive agent.
 13. The method of claim 12, wherein said composition is inthe form of a sheet material.
 14. The method of claim 12, wherein saidbiodegradable polymeric film comprises a polymer selected from the groupconsisting of polylactide, polyglycolide, poly lactide-glycolidecopolymers, and alginate.
 15. The method of claim 12, wherein said atleast one additional active agent is selected from the group consistingof antibiotics, growth factors, steroids, and antineoplastic agents. 16.The method of claim 12, wherein said peroxide is calcium peroxide. 17.The method claim 12, wherein said peroxide is sodium percarbonate.